基于eDNA宏条形码技术的喀斯特高原人工湖泊鱼类多样性分析

【目的】利用eDNA宏条形码技术探究喀斯特高原人工湖泊鱼类多样性，并综合评估环境因子对鱼类分布的影响，为喀斯特地区水域鱼类多样性评估提供新方法、新思路，也为红枫湖和阿哈湖鱼类保护管理措施的制定积累基础资料。【方法】以红枫湖和阿哈湖为代表采集水样，以Tele 02硬骨鱼类通用引物PCR扩增eDNA，在Illumina NovaSeq 6000测序平台完成高通量测序；对优质序列按照98%相似性进行OTU聚类，生成OTUs表格，通过基因注释获得鱼类物种信息，并以R语言分别进行PerMANOVA分析和冗余分析（RDA）。【结果】在红枫湖鉴定出鱼类32种，隶属于5目11科27属，其中鲤形目鱼类18种，红枫湖南、北湖鱼类群落结构差异不显著（R2=0.08，P=0.562>0.05）；在阿哈湖鉴定到鱼类33种，隶属于6目12科28属，其中鲤形目鱼类18种，各采样点检出鱼类总数差异不明显，保持在28~32种。现阶段的红枫湖和阿哈湖鱼类组成以鲤形目鱼类为主，但存在以日鲈目为主的养殖鱼类生态入侵。红枫湖环境因子与鱼类群落结构差异的决定系数（R2）排序为溶解氧（DO）>总溶解固体（TDS）=水温（WT）>pH>离子氨（NH₄+），DO（P=0.034）是引起红枫湖各采样点鱼类群落结构差异的主要环境因子；阿哈湖环境因子与鱼类群落结构差异的决定系数（R2）排序为TDS>WT>Tra>NH₄+>DO，TDS（P=0.005）是引起阿哈湖各采样点鱼类群落结构差异的主要环境因子。【结论】现阶段的红枫湖和阿哈湖鱼类组成以鲤形目鱼类为主，但存在以日鲈目为主的养殖鱼类生态入侵。eDNA宏条形码技术在喀斯特高原水域鱼类多样性评估方面具有较好的适应性，可快速监测到鱼类群落结构和空间分布，但通用引物可能对某些鱼类存在扩增偏好性；此外，不同人工湖泊水质理化因子特异性较强，研究环境因子与湖泊鱼类群落的关系时应对每个湖泊单独进行评估。

Fish diversity analysis of artificial lakes in karst plateau based on eDNA metabarcoding technology

【Objective】 To investigate fish diversity of artificial lakes in karst plateau using eDNA metabarcoding technology and to comprehensively assess effects of environmental factors on fish distribution, so as to provide new methods and perspectives for fish diversity assessment in waters of karst area and accumulate basic information for the formation of fish conservation management measures in Hongfeng Lake and Aha Lake.【Method】 Water collected from Hongfeng Lake and Aha Lake were taken as representative samples. eDNA was amplified by PCR with Tele 02 universal primers for teleost fish and high-throughput sequencing was completed on Illumina NovaSeq 6000 sequencing platform;OTU clustering was performed and OTUs tables were generated for high-quality sequences according to 98% similarity, and then fish species information was obtained by gene annotation. PerMANOVA analysis and redundancy analysis(RDA) were performed with R language.【Result】 Thirty-two species of fish were identified in Hongfeng Lake, belonging to 27 genus, 11 families of 5 orders, including 18 species of Cypriniformes fishes, and the difference in fish community structure between the south and north lakes of Hongfeng Lake was not significant(R2=0.08, P=0.562>0.05);33 species of fish were identified in Aha Lake, belonging to 28 genus, 12 families of 6 orders, including 18 species of Cypriniformes fishes, and the difference in the total number of fish detected at each sampling site was not significant. The total number of fish detec-ted at each sampling site did not vary significantly, remaining at 28-32 species. At present, the fish composition of Hongfeng Lake and Aha Lake was mainly Cypriniformes fish, but an ecological invasion of farmed fish, mainly Centrarchiformes fishes was found. Determination coefficient(R2) of the differences between environmental factors and fish community structure in Hongfeng Lake was dissolved oxygen(DO) >total dissolved solids(TDS)=water temperature(WT) >pH>ionic ammonia(NH₄+), and DO(P=0.034) was the main environmental factor causing the differences in fish community structure at each sampling site in Hongfeng Lake;determination coefficient(R2) of the differences between environmental factors and fish community structure in Aha Lake was TDS>WT>Tra>NH₄++>DO, and TDS(P=0.005) was the main environmental factor causing the difference in fish community structure among the sampling sites in Aha Lake.【Conclusion】 At present, the fish composition of Hongfeng Lake and Aha Lake is dominated by Cypriniformes fishes, but an ecological invasion of farmed fish, mainly Centrarchiformes fishes is found. eDNA metabarcoding technology has excellent adaptability in karst plateau waters fish diversity evaluation, thus it can quickly monitor the fish community structure and spatial distribution, but universal primers may have amplification preference for some fish;in addition, different artificial lakes are more specific in terms of water physical and chemical factors, therefore, relationship between environmental factors and lake fish communities should be evaluated separately for each lake.